Format

Send to

Choose Destination
Proc Natl Acad Sci U S A. 2017 May 9;114(19):4954-4959. doi: 10.1073/pnas.1615585114. Epub 2017 Apr 24.

Biochemical and structural characterization of oxygen-sensitive 2-thiouridine synthesis catalyzed by an iron-sulfur protein TtuA.

Author information

1
Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan.
2
Japan Biological Information Research Center, Japan Biological Informatics Consortium (JBIC), Tokyo 135-0064, Japan.
3
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan.
4
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan.
5
Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo 135-0064, Japan.
6
Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan.
7
Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo 135-0064, Japan; naoki-shigi@aist.go.jp tanaka@sci.hokudai.ac.jp.
8
Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan; naoki-shigi@aist.go.jp tanaka@sci.hokudai.ac.jp.
9
Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Sapporo 060-0810, Japan.

Abstract

Two-thiouridine (s2U) at position 54 of transfer RNA (tRNA) is a posttranscriptional modification that enables thermophilic bacteria to survive in high-temperature environments. s2U is produced by the combined action of two proteins, 2-thiouridine synthetase TtuA and 2-thiouridine synthesis sulfur carrier protein TtuB, which act as a sulfur (S) transfer enzyme and a ubiquitin-like S donor, respectively. Despite the accumulation of biochemical data in vivo, the enzymatic activity by TtuA/TtuB has rarely been observed in vitro, which has hindered examination of the molecular mechanism of S transfer. Here we demonstrate by spectroscopic, biochemical, and crystal structure analyses that TtuA requires oxygen-labile [4Fe-4S]-type iron (Fe)-S clusters for its enzymatic activity, which explains the previously observed inactivation of this enzyme in vitro. The [4Fe-4S] cluster was coordinated by three highly conserved cysteine residues, and one of the Fe atoms was exposed to the active site. Furthermore, the crystal structure of the TtuA-TtuB complex was determined at a resolution of 2.5 Å, which clearly shows the S transfer of TtuB to tRNA using its C-terminal thiocarboxylate group. The active site of TtuA is connected to the outside by two channels, one occupied by TtuB and the other used for tRNA binding. Based on these observations, we propose a molecular mechanism of S transfer by TtuA using the ubiquitin-like S donor and the [4Fe-4S] cluster.

KEYWORDS:

2-thiouridine; Fe-S cluster; crystal structure; sulfur transfer; tRNA modification

PMID:
28439027
PMCID:
PMC5441745
DOI:
10.1073/pnas.1615585114
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center